The phospholipid bilayer surrounding animal cells is a dynamic environment made up of four principle phospholipid components, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). These four phospholipids are distributed between two monolayers of the membrane in an asymmetrical fashion, with the choline-containing lipids, PC and SM, largely populating the external leaflet, while the aminophospholipids, PE and especially PS, are restricted primarily to the inner membrane leaflet. This membrane asymmetry has been known for some time, and there is a consensus that it is maintained by the concerted action of a family of translocase enzymes. Efforts to elucidate the structure and mechanism of these transport proteins are ongoing and are described elsewhere.
Apoptosis, or the sequence of events collectively known as “programmed cell death,” is an important process whereby cells are intentionally marked for clearance from the body. Apoptosis is a central process in developmental biology and also in many types of diseases. For example, selective induction of apoptosis in cancerous tissue is an attractive chemotherapeutic strategy, and detection of apoptosis is therefore a key step in the drug development process. Various strategies for detecting apoptosis have been reported, including monitoring of intracellular caspase activity, observing nucleic acid fragmentation, and detection of membrane permeabilization. These assays are employed as diagnostic tools for identifying apoptosis, but each has limitations that render it imperfect in certain situations.
Loss of the phospholipid asymmetry inherent to healthy animal cell membranes is a hallmark of apoptosis, regardless of the initiating stimulus. During the early to middle stages of apoptosis, the PS normally found exclusively on the inner membrane monolayer becomes scrambled between the two membrane leaflets. PS is the most abundant anionic phospholipid component in the plasma membrane of most animal cells, and PS externalization is a contributing factor to the recognition of dead and dying cells by macrophages. The externalized PS can be detected on the cell surface using indicator-labeled reagents that preferentially bind the PS headgroup. PS externalization precedes the upregulation of protease activity in the cytosol, and occurs before membrane permeabilization begins. Another attractive feature of this cell surface assay is that it avoids the complications of other assays that require access to the cytosol. Furthermore, there is evidence that PS exposure on the cell surface is a common final outcome for other death processes such as senescence, mitotic catastrophe and autophagy, etc. Thus, the strategy of PS recognition makes it possible to consider applications for site-specific in vivo imaging of dead and dying tissue that would be useful in the treatment of various diseases such as cancer and cardiovascular disease.
The annexins are a group of proteins that bind anionic phospholipids in a Ca2+-dependent manner. One member of the family, Annexin V (Anx V), binds PS with high selectivity and high affinity in the presence of Ca2+, making it well suited for detection of apoptosis. A variety of fluorophore-labeled versions of Anx V are commercially available, and detection of cell-surface PS by this technique has become a standard protocol in cell biology research.
Even though Anx V derivatives are widely used for PS-sensing applications and apoptosis detection, Anx V has several disadvantages and limitations. For instance, the unfunctionalized Anx V protein has a mass of about 36 KDa, which restricts its use to those applications where a PS sensor of this size can be accommodated. Furthermore, Anx V-PS binding requires millimolar levels of Ca2+ in order to produce the nanomolar dissociation constants that make using the protein desirable. This level of Ca2+ may be problematic in situations where other processes may need to be monitored simultaneously. Additionally, animal cells frequently have integral membrane phospholipid transport proteins, called “scramblases,” that can move phospholipids nonspecifically between the two membrane monolayers. These scramblases are activated by micromolar Ca2+ levels, well below that necessary for Anx V-PS binding. Thus, false positives may occur when using Anx V to detect apoptosis. The rate of Anx V-PS binding is also quite slow. Complete membrane binding by Anx V-PS often requires incubation periods of up to one hour, which is problematic for many types of kinetic assays: Anx V is also susceptible to N-terminal proteolytic degradation. In addition, annexin V is a protein that may not have the necessary chemical stability for employment in high-throughput screening of cancer drugs, and may lack the biochemical stability necessary for in vivo imaging of dying tissue.
Another report provided an anthracene-derivated DPA zinc complex for sensing apoptotic cells. However, anthracene is often not an ideal probe in imaging studies because of the short emission wavelength and photobleaching.
These and other limitations demonstrate that a need continues to exist in the art for alternative molecular probes that may be substituted for annexin V, that will bind PS-rich membranes in a Ca2+-independent manner. These kinds of molecules would be extremely useful in further characterizing, detecting, monitoring and/or screening for cell apoptosis and other clinical conditions associated with a relative increase and/or presence of PS.
In addition, the anionic surface of bacterial cells provides an environment that is analogous in certain characteristics with apoptotic cells. In particular, the surfaces of bacterial cells are anionic and thus probes targeting anionic cell surfaces may be used to identify the presence of bacteria. Thus, a suitable molecular probe or group of probes may be capable both of detecting the presence of apoptotic cells and the presence of bacterial cells.